Various methods have been reported for induced differentiation of human pluripotent stem cells into cardiomyocytes and include, for example, a method in which human pluripotent stem cells maintained on feeder cells are differentiated with activin A and BMP4 (bone morphogenetic protein 4) (Non Patent Literature 1 and 2), a method in which human pluripotent stem, cells maintained on Matrigel are differentiated with Wnt3a, R-Spondin-1 and DKK1 (Non Patent Literature 3), and a method in which human pluripotent stem cells maintained on feeder cells are differentiated on a large scale in a bioreactor (Non Patent Literature 3).
When pluripotent stem cells are induced to differentiate into cardiomyocytes, the differentiation efficiency usually varies from lot to lot. In the case where such cardiomyocytes are used as a cell source for transplantation in regenerative medicine, if not meeting the purity criteria, they need to be purified. The ideal purity of human pluripotent stem cell-derived cardiomyocytes for use in regenerative medicine is not necessarily 100%, and in fact, about 70% purity is better from a functional perspective according to the previous report (Non Patent Literature 4). Therefore, if human pluripotent stem cell-derived cardiomyocytes with a purity of less than 70% can be enriched, to a purity of about 70%, it will be of great significance in particular in the preparation of a cell source for transplantation in cell therapy. In addition, since it is difficult to achieve the same level of the efficiency of cardiomyocyte differentiation in every lot, the differences in the efficiency among lots probably affect the reproducibility of the results in new drug screening studies. Moreover, sensitive detection of the drug response of cardiomyocytes requires the minimization of the background noise from the cells other than cardiomyocytes.
Several methods have already been reported for the increase of the purity of cardiomyocytes after induced differentiation. These methods can be roughly classified into two categories. The methods in the first category utilize the cell's auxotrophy to eliminate cells other than cardiomyocytes. That is, they are methods in which a medium deprived of nutrients essential for the survival of cells other than cardiomyocytes is used for culture to eliminate these cells. For example, Patent Literature 1 describes a method for increasing the purity of cardiomyocytes, the method comprising using a medium with a low glucose concentration. However, this method causes great damage to cardiomyocytes in addition to other cells, which is matter of concern. Another problem is that the selection of cardiomyocytes requires a prolonged time (at least a few days).
The methods in the second category utilize antibodies against cardiomyocyte-specific surface antigens or glycans to isolate cardiomyocytes. That is, they are methods using flow cytometry or magnetic beads. However, disadvantageously, in the case of using flow cytometry, the time required for cell isolation significantly increases in accordance with the increase in the required amount of the cells. In addition, the use of flow cytometry and magnetic beads cause a concern about the damage and contamination of the retrieved, cells. Therefore, it is not realistic to employ these methods in the case requiring a large quantity of cells, for example, in the case of cell transplantation. Moreover, in these methods, the examination of whether or not residual antibodies or magnetic beads are present is essential and takes time and effort. If residual magnetic beads are detected in the isolated cells, the safety of the cells for transplantation is difficult to guarantee.
An ideal method for preparing cells to be used for transplantation through the purification of differentiated cells derived from human pluripotent stem cells needs to meet the following requirements: the purity and yield of differentiated cells is high; the safety for human recipients is guaranteed; and the prepared cells are capable of performing the desired functions in vivo after transplantation. More ideally, the preparation method can handle a large quantity of cells in a short period of time in a simple manner with causing less damage to the cells. However, in reality, previously reported methods are unsatisfactory for the preparation of clinically applicable cells. Therefore, strongly desired is the development of a novel method for preparing a large quantity of clinically applicable cells, i.e., safe and less damaged cells, in a simple manner.